Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04-May-2011 to 23-May-2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
The study integrity was not adversely affected by the deviations.
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Yellow powder (determined at NOTOX)
Specific details on test material used for the study:
Test substance storage At room temperature in the dark Stability under storage conditions Stable Expiry date 15 April 2012 (allocated by NOTOX, 1 year after receipt of the test substance)

In vivo test system

Test animals

Species:
mouse
Strain:
CBA:J
Sex:
female
Details on test animals and environmental conditions:
Conditions Animals were housed in a controlled environment, in which optimal conditions were considered to be approximately 15 air changes per hour, a temperature of 21.0 ± 3.0ºC (actual range: 21.0 – 23.6ºC), a relative humidity of 40-70% (actual range: 35 - 70%) and 12 hours artificial fluorescent light and 12 hours darkness per day.

Accommodation Animals were group housed in labeled Makrolon cages (MIII type; height 18 cm) containing sterilised sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France). Paper (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) and shelters (disposable paper corner home, MCORN 404, Datesand Ltd, USA) were supplied as cage-enrichment. The acclimatization period was at least 5 days before the start of treatment under laboratory conditions. On Day 6, the animals were group housed in Makrolon MII type cages with a sheet of paper instead of sawdust and cage enrichment. Certificates of analysis were examined and then retained in the NOTOX archives.

Diet Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).

Water Free access to tap water.

Results of analysis for diet (nutrients and contaminants), sawdust, paper and water were assessed and did not reveal any findings that were considered to have affected the study integrity. All certificates and results of analysis are retained in the NOTOX archives.

Weight of evidence analysis
In the interest of animal welfare and to minimize any testing likely to produce severe responses in animals, a weight of evidence analysis was performed prior to start of this study. All available information was evaluated (e.g. existing human and animal data, literature, substance data supplied by the sponsor, analysis of structure activity relationships (SAR), physicochemical properties and reactivity (pH, buffering capacity).

Pre-screen test
A pre-screen test was conducted in order to select the highest test substance concentration to be used in the main study. In principle, this highest concentration should cause no systemic toxicity, may give moderate irritation at the most (maximum grade 2 (see section 6.7) and/or an increase in ear thickness < 25%) and is the highest possible concentration that can technically be applied.

Two test substance concentrations were tested; a 25% and 50% concentration. The highest concentration was the maximum concentration as required in the test guidelines (undiluted for liquids, 50% for solids).

The test system, procedures and techniques were identical to those used in the main study except that assessment of lymph node proliferation and necropsy were not performed. Two young adult animals per concentration were selected (in the range of 8 to14 weeks old). Each animal was treated with one concentration on three consecutive days. Animals were group housed in labeled Makrolon cages (MII type, height 12.5 cm). Ear thickness measurements were conducted using a digital thickness gauge prior to dosing on Days 1 and 3, and on Day 6. Since test substance remnants were anticipated to hamper scoring after dosing on Days 2 and 3 (based on Day 1 observations), an additional scoring of the ears was conducted prior to dosing on Days 2 and 3, next to scoring of the ears after dosing. Animals were sacrificed after the final observation.

Main study
Three groups of five animals were treated with one test substance concentration per group. The highest test substance concentration was selected from the pre-screen test. One group of five animals was treated with vehicle.

Induction - Days 1, 2 and 3
The dorsal surface of both ears was topically treated (25 μL/ear) with the test substance concentration, at approximately the same time on each day. The concentrations were stirred with a magnetic stirrer immediately prior to dosing.

The control animals were treated in the same way as the experimental animals, except that the vehicle was administered instead of the test substance.
Excision of the nodes - Day 6
All animals: Each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline (PBS) (Merck, Darmstadt, Germany) containing 20 μCi of 3H-methyl thymidine (PerkinElmer Life and Analytical Sciences, Boston, MA, US).

After approximately five hours, all animals were killed by intraperitoneal injection (0.2 mL/animal) with Euthasol® 20% (AST Farma BV, Oudewater, The Netherlands). The draining (auricular) lymph node of each ear was excised. The relative size of the nodes (as compared to normal) was estimated by visual examination and abnormalities of the nodes and surrounding area were recorded. The nodes were pooled for each animal in approximately 3 mL PBS.

Tissue processing for radioactivity - Day 6
A single cell suspension of lymph node cells (LNC) was prepared in PBS by gentle separation through stainless steel gauze (diameter 125 μm). LNC were washed twice with an excess of PBS by centrifugation at 200g for 10 minutes at 4ºC. To precipitate the DNA, the LNC were exposed to 5% trichloroacetic acid (TCA) (Merck, Darmstadt, Germany) and stored in the refrigerator until the next day.

Radioactivity measurements - Day 7
Precipitates were recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL of Ultima Gold cocktail (PerkinElmer Life and Analytical Sciences, Boston, MA, US) as the scintillation fluid. Radioactive measurements were performed using a Packard scintillation counter (2800TR). Counting time was to a statistical precision of ± 0.2% or a maximum of 5 minutes whichever came first. The scintillation counter was programmed to automatically subtract background and convert Counts Per Minute (CPM) to Disintegrations Per Minute (DPM).

Observations
Mortality/Viability Twice daily.

Body weights On Day 1 (pre-dose) and Day 6 (prior to necropsy).

Clinical signs Once daily on Days 1-6 (on Days 1-3 between 3 and 4 hours after dosing).

Irritation Once daily on Days 1-6 (on Days 1 - 3 immediately after dosing) according to the following numerical scoring system. Furthermore, a description of all other (local) effects was recorded.

Interpretation
DPM values are presented for each animal and for each dose group. A Stimulation Index (SI) is calculated for each group. The SI is the ratio of the DPM/group compared to DPM/vehicle control group.

If the results indicate a SI ≥ 3, the test substance may be regarded as a skin sensitizer, based on the test guideline and recommendations done by ICCVAM.

The results were evaluated according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2007) and the Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008 on classification, labelling and packaging of substances and mixtures.

The EC3 value (the estimated test substance concentration that will give a SI =3) was determined, using linear interpolation.

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
test substance concentrations of 10, 25 or 50% w/w
No. of animals per dose:
5f/group
Details on study design:
Pre-screen test
A pre-screen test was conducted in order to select the highest test substance concentration to be used in the main study. In principle, this highest concentration should cause no systemic toxicity, may give moderate irritation at the most (maximum grade 2 (see section 6.7) and/or an increase in ear thickness < 25%) and is the highest possible concentration that can technically be applied.

Two test substance concentrations were tested; a 25% and 50% concentration. The highest concentration was the maximum concentration as required in the test guidelines (undiluted for liquids, 50% for solids).

The test system, procedures and techniques were identical to those used in the main study except that assessment of lymph node proliferation and necropsy were not performed. Two young adult animals per concentration were selected (in the range of 8 to14 weeks old). Each animal was treated with one concentration on three consecutive days. Animals were group housed in labeled Makrolon cages (MII type, height 12.5 cm). Ear thickness measurements were conducted using a digital thickness gauge prior to dosing on Days 1 and 3, and on Day 6. Since test substance remnants were anticipated to hamper scoring after dosing on Days 2 and 3 (based on Day 1 observations), an additional scoring of the ears was conducted prior to dosing on Days 2 and 3, next to scoring of the ears after dosing. Animals were sacrificed after the final observation.

Main study
Three groups of five animals were treated with one test substance concentration per group. The highest test substance concentration was selected from the pre-screen test. One group of five animals was treated with vehicle.

Vehicle Dimethyl sulphoxide (Merck, Darmstadt, Germany). Rationale The vehicle was selected based on trial formulations performed at NOTOX and on test substance data supplied by the sponsor. Preparation The test substance formulations (w/w) were prepared within 4 hours prior to each treatment. No adjustment was made for specific gravity of the vehicle. Homogeneity was obtained to visually acceptable levels.
Positive control substance(s):
other: no concurrent positive control group was added to the study.
Statistics:
Radioactivity measurements- Day 7
Counting time was to a statistical precision of ± 0.2% or a maximum of 5 minutes whichever came first.

Interpretation
If the results indicate a SI ≥ 3, the test substance should be regarded as a skin sensitizer. In case of borderline results, statistical analysis may be performed to determine the dose response relationship and pair wise comparisons between dose groups versus negative control.

Results and discussion

Positive control results:
no concurrent positive control group was added to the study.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
ca. 2.4
Test group / Remarks:
10 %
Key result
Parameter:
SI
Value:
ca. 3.1
Test group / Remarks:
25 %
Key result
Parameter:
SI
Value:
ca. 9.7
Test group / Remarks:
50 %
Cellular proliferation data / Observations:
Interpretation
DPM values will be presented for each animal and for each dose group. A Stimulation Index (SI) will be calculated for each group. The SI is the ratio of the DPM/group compared to DPM/vehicle control group.

If the results indicate a SI ≥ 3, the test substance should be regarded as a skin sensitizer. In case of borderline results, statistical analysis may be performed to determine the dose response relationship and pair wise comparisons between dose groups versus negative control. The results can be evaluated according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations and the Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008 on classification, labelling and packaging of substances and mixtures.

The EC3 value (the estimated test substance concentration that will give a SI=3) may be determined if possible, based on the dose response relationship or calculated using Linear Interpolation (reference 1).

If it is not possible to determine the SI=3 value, additional groups of animals may be treated. This will be done in consultation with the sponsor and will be confirmed by protocol amendment

CLINICAL OBSERVATIONS:
No mortality occurred and no symptoms of systemic toxicity were observed in the animals of the main study.

Skin reactions / Irritation
Brown remnants of the test substance were present on both ears of all animals at 10, 25 and 50% between Days 1 and 6, due to which erythema could not be scored between Days 1 and 4 (10 and 25%) or between Days 1 and 5 (50%). No oedema was present.

Macroscopy of the auricular lymph nodes and surrounding area
All auricular lymph nodes at 50% and the majority of auricular lymph nodes at 10 and 25% appeared larger in size when compared to the control group. The largest auricular lymph nodes were found at 50%. No macroscopic abnormalities of the surrounding area were noted in any of the animals.

Radioactivity measurements
Mean DPM/animal values for the experimental groups treated with test substance concentrations 10, 25 and 50% were 1253, 1602 and 5024 DPM respectively. The mean DPM/animal value for the vehicle control group was 517 DPM.

BODY WEIGHTS
Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

Applicant's summary and conclusion

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
The SI values calculated for the substance concentrations 10, 25 and 50% were 2.4, 3.1 and 9.7 respectively.

These results indicate that the test substance could elicit an SI ≥ 3. The data showed a dose-response and an EC3 value (the estimated test substance concentration that will give a SI =3) of 22.9% was calculated.

The six-month reliability check with Alpha-hexylcinnamicaldehyde indicates that the Local Lymph Node Assay as performed at NOTOX is an appropriate model for testing for contact hypersensitivity (see Appendix 1). Based on these results: - according to the recommendations made in the test guidelines, 9-Aminominocycline would be regarded as skin sensitizer.

- according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2007), 9-Aminominocycline should be classified as skin sensitizer (Category 1).

- according to the Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures, 9-Aminominocycline should be classified as skin sensitizer (Category 1) and labeled as H317: May cause an allergic skin reaction.
Executive summary:

Assessment of Contact Hypersensitivity to 9-Aminominocycline in the Mouse (Local Lymph Node Assay).  

The study was carried out based on the guidelines described in:  OECD, Section 4, Health Effects, No.429 (2010),  EC, No 440/2008; B42: "Skin Sensitization: Local Lymph Node Assay" EPA, OPPTS 870.2600 (2003) “Skin Sensitization”.

Test substance concentrations selected for the main study were based on the results of a pre-screen test.

In the main study, three experimental groups of five female CBA/J mice were treated with test substance concentrations of 10, 25 or 50% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with vehicle alone (Dimethyl sulphoxide).

Three days after the last exposure, all animals were injected with 3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised and pooled for each animal. After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. The activity was expressed as the number of Disintegrations Per Minute (DPM) and a stimulation index (SI) was subsequently calculated for each group.

Brown remnants of the test substance were present on both ears of all animals at 10, 25 and 50% between Days 1 and 6, due to which erythema could not be scored between Days 1 and 4 (10 and 25%) or between Days 1 and 5 (50%). No oedema was present.

All auricular lymph nodes at 50% and the majority of auricular lymph nodes at 10 and 25% appeared larger in size when compared to the control group. The largest auricular lymph nodes were found at 50%. No macroscopic abnormalities of the surrounding area were noted in any of the animals.

Mean DPM/animal values for the experimental groups treated with test substance concentrations 10, 25 and 50% were 1253, 1602 and 5024 DPM respectively. The mean DPM/animal value for the vehicle control group was 517 DPM.

The SI values calculated for the substance concentrations 10, 25 and 50% were 2.4, 3.1 and 9.7 respectively.

These results indicate that the test substance could elicit an SI ≥ 3. The data showed a dose-response and an EC3 value (the estimated test substance concentration that will give a SI =3) of 22.9% was calculated.

The six-month reliability check with Alpha-hexylcinnamicaldehyde indicates that the Local Lymph Node Assay as performed at NOTOX is an appropriate model for testing for contact hypersensitivity.   Based on these results: − according to the recommendations made in the test guidelines, 9-Aminominocycline would be regarded as skin sensitizer. − according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2007), 9-Aminominocycline should be classified as skin sensitizer (Category 1). − according to the Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures, 9-Aminominocycline should be classified as skin sensitizer (Category 1) and labeled as H317: May cause an allergic skin reaction.